Discovery of small-molecule modulator of heterotrimeric G_i-protein by integrated phenotypic profiling and chemical proteomics

Discovery of small-molecule inducers of unique phenotypic changes combined with subsequent target identification often provides new insights into cellular functions. Here, we applied integrated profiling based on cellular morphological and proteomic changes to compound screening. We identified an indane derivative, NPD9055, which is mechanistically distinct from reference compounds with known modes of action. Employing a chemical proteomics approach, we then showed that NPD9055 binds subunits of heterotrimeric G-protein G_i. An in vitro [³⁵S]GTPγS-binding assay revealed that NPD9055 inhibited GDP/GTP exchange on a Gα_i subunit induced by a G-protein-coupled receptor agonist, but not on another G-protein from the Gα_s family. In intact HeLa cells, NPD9055 induced an increase in intracellular Ca²⁺ levels and ERK/MAPK phosphorylation, both of which are regulated by Gβγ, following its dissociation from Gα_i. Our observations suggest that NPD9055 targets Gα_i and thus regulates Gβγ-dependent cellular processes, most likely by causing the dissociation of Gβγ from Gα_i.